Thiolactones and Δ8,9-Pregnene Steroids from the Marine-Derived Fungus Meira sp. 1210CH-42 and Their α-Glucosidase Inhibitory Activity

The fungal genus Meira was first reported in 2003 and has mostly been found on land. This is the first report of second metabolites from the marine-derived yeast-like fungus Meira sp. One new thiolactone (1), along with one revised thiolactone (2), two new Δ8,9-steroids (4, 5), and one known Δ8,9-steroid (3), were isolated from the Meira sp. 1210CH-42. Their structures were elucidated based on the comprehensive spectroscopic data analysis of 1D, 2D NMR, HR-ESIMS, ECD calculations, and the pyridine-induced deshielding effect. The structure of 5 was confirmed by oxidation of 4 to semisynthetic 5. In the α-glucosidase inhibition assay, compounds 2–4 showed potent in vitro inhibitory activity with IC50 values of 148.4, 279.7, and 86.0 μM, respectively. Compounds 2–4 exhibited superior activity as compared to acarbose (IC50 = 418.9 μM).


Introduction
Fungi constitute one of the largest groups of organisms. Fungal-derived natural products (NPs) are pharmaceutically abundant, with several important biological applications ranging from highly potent toxins to approved drugs [1]. In particular, secondary metabolites obtained from marine fungi have garnered significant interest due to their unique chemical structures and potential biomedical applications [1,2]. While the number of cultivable marine fungi is extremely low (1% or less) compared to their global biodiversity [1,3], more than 1000 molecules have been reported and characterized from marine fungi, including alkaloids, lipids, peptides, polyketides, prenylated polyketides, and terpenoids [4][5][6][7]. Most research on secondary metabolites produced by marine fungi has primarily focused on a few genera, including Penicillium, Aspergillus, Fusarium, and Cladosporium [8,9]. Research into natural products derived from marine fungi is continually expanding, and as a result, a broader range of genera is now being investigated, with a particular focus on those associated with unique substrates and previously unexplored habitats [10][11][12].
In 2003, the genus Meira was first reported, namely M. geulakonigii and M. argovae, as a novel basidiomycetous [13]. M. geulakonigii was isolated from the citrus rust mite on pummelo (Citrus grandis), and M. argovae originated from a carmine spider mite on the leaves of castor bean (Ricinus communis) [8]. These Meira species have a similar morphology to yeast-like fungi. Nonetheless, the phylogenetic analysis of rDNA sequence data has identified Meira as a member of the Brachybasidiaceae family within the Exobasidiales, which has identified Meira as a member of the Brachybasidiaceae family within the Exobasidiales, which is classified under the Ustilaginomycetes (Basidiomycota) in the Exobasidiomycetidae group. [14]. M. geulakonigii has been used successfully as a biological control agent against citrus and other phytophagous mites, as well as powdery mildew fungi. [13,[15][16][17]. A potential biocontrol agent against five mite species has been demonstrated for M. argovae [18]. Recently, M. nicotianae came from the rhizosphere of tobacco root, and that strain has the capability to promote plant growth possible in similar ways as plant growth-promoting fungi and arbuscular mycorrhizal fungi [19].
In this study, we isolated a yeast-like fungal species from a seawater sample. Phylogenetic analysis of ITS rDNA indicated that strain 1210CH-42 is closely related to other Meira species: Meira sp. M40, M. nashicola CY-1, and M. miltonrushii NIOSN-SK46-S121. So far, there are only a few reports on the isolation of Meira strains, but natural products from the genus Meira have not been investigated. This is the first report on the secondary metabolites from the marine-derived yeast-like fungus Meira. Herein, we report the isolation, structure elucidation, α-glucosidase inhibitory activity of 1-5, and the structure revision of 2 isolated from the Meira strain 1210CH-42 ( Figure 1).
Detailed analysis of 3 J H,H coupling constants and 1D NOESY data determined the relative configuration of 1. The relative stereochemistry of C-2 could be established by the observation of strong selective 1D NOESY correlations between H-2 and H-3/H-4b, between H-4b and H-2/H-3, and between H-5 and H-4a ( Figure 2). These correlations suggested that the relative configurations of C-2 and C-3 must be cis rather than transconfiguration in 1. Thus, the relative configuration of 1 could be assigned as 2S*, 3R*. To determine the absolute configuration of 1, the theoretical electronic circular dichroism (ECD) spectra of 1 and its enantiomer were calculated. The experimental ECD spectrum of 1 showed a good agreement with the calculated ECD spectrum of the 2S, 3R-isomer ( Figure 3). Therefore, the structure of 1 was elucidated to be a 2S-acetamide-3R-methyl-thiolactone.   Detailed analysis of 3 JH,H coupling constants and 1D NOESY data de ative configuration of 1. The relative stereochemistry of C-2 could be es observation of strong selective 1D NOESY correlations between H-2 an tween H-4b and H-2/H-3, and between H-5 and H-4a ( Figure 2). These gested that the relative configurations of C-2 and C-3 must be cis rather t uration in 1. Thus, the relative configuration of 1 could be assigned as 2S mine the absolute configuration of 1, the theoretical electronic circular spectra of 1 and its enantiomer were calculated. The experimental EC showed a good agreement with the calculated ECD spectrum of the 2S, 3 3). Therefore, the structure of 1 was elucidated to be a 2S-acetamide lactone.  Detailed analysis of 3 JH,H coupling constants and 1D NOESY data determined the relative configuration of 1. The relative stereochemistry of C-2 could be established by the observation of strong selective 1D NOESY correlations between H-2 and H-3/H-4b, between H-4b and H-2/H-3, and between H-5 and H-4a ( Figure 2). These correlations suggested that the relative configurations of C-2 and C-3 must be cis rather than trans-configuration in 1. Thus, the relative configuration of 1 could be assigned as 2S*, 3R*. To determine the absolute configuration of 1, the theoretical electronic circular dichroism (ECD) spectra of 1 and its enantiomer were calculated. The experimental ECD spectrum of 1 showed a good agreement with the calculated ECD spectrum of the 2S, 3R-isomer ( Figure  3). Therefore, the structure of 1 was elucidated to be a 2S-acetamide-3R-methyl-thiolactone.  Compound 2 was isolated as a white amorphous powder. The molecular formula of 2 was the same as that of 1 (C 7 H 11 NO 2 S) based on the HR-ESIMS data. Furthermore, Mar. Drugs 2023, 21, 246 4 of 11 the 1D NMR data of 2 were also similar but not identical to those of 1 ( Table 1). The planar structure of 2 was determined to be the same as 1 by analysis of 1 H-1 H COSY and HMBC data ( Figure 2). However, the 1 H and 13 C chemical shifts of 2 were different from 1, especially those for the chiral centers, suggesting that the stereochemistry of 2 might be different from 1. The relative configuration of 2 was also determined by analysis of 3 J H,H coupling constants and selective 1D NOESY data. The relative stereochemistry of C-2 could be established through the observation of strong NOESY contacts between H-2 and H-4a/H-5, between H-4a and H-2/H-5, and between H-4b and H-3. A relatively large coupling constant was observed between H-2 and H-3 ( 3 J H,H = 12.5 Hz). Thus, the relative configurations of H-2 and H-3 had a trans-configuration in 2 ( Figure 2). The J-based configurational analysis and NOESY measurements could not discriminate the possible relative configurations for (2S*, 3S*) or (2R*, 3R*). To solve this issue and to determine the absolute configuration of 2, the ECD spectra of 2 and its enantiomer were calculated. The experimental ECD spectrum of 2 showed a good agreement with the calculated ECD spectrum of the 2R, 3R-isomer ( Figure 3). Therefore, the structure of 2 was elucidated as an epimer of 1 and to be a 2R-acetamide-3R-methyl-thiolactone.
Notably, the 1 H and 13 C NMR data in CDCl 3 of 2 were almost the same as those of the previously reported thiolactone with 2R, 3S-configuration isolated from a Penicillium chrysogenum (Table S1 and Figure S15) [20]. The reported compound with 2R, 3S-configuration possesses the same planar structure as 2 in this study. In the original paper for the compound with 2R, 3S-configuration, by the NOE correlation between H-3 (δ H 2.24) and H-2 (δ H 4.45), the authors insisted that the two protons were oriented on the same side of the ring system. However, its 1D NOE spectrum for the reported compound showed signals from H-3 (δ H 2.24) to H-2/H-4/H-5/H-6 and NH, making it unclear to determine the orientation of H-3 to the same side of H-2 or not ( Figures S15 and S16). Moreover, if the reported configuration is correct, H-2 and H-3 are in syn relation, and they should have a small scalar coupling constant, but H-2 in the reported thiolactone had a large coupling constant (12.5 Hz) as in the revised structure (Table S1). In this study, we carefully compared and checked the selective 1D NOESY data of 2 with those for the reported compound. As noted above, 2 exhibited strong NOE correlations from H-2 to H-5/ H-4a and from H-4b to H-3 but not from H-4b to H-2, suggesting that H-2 and H-5 are on the same face. Furthermore, the reported compound with 2R, 3S-configuration and 1 (2S, 3R-configuration) are enantiomers and should have the same but opposite-in-sign specific rotation values. However, the optical rotation values of the reported thiolactone and 1 were be established through the observation of strong NOESY contacts be 4a/H-5, between H-4a and H-2/H-5, and between H-4b and H-3. A r pling constant was observed between H-2 and H-3 ( 3 JH,H = 12.5 Hz) configurations of H-2 and H-3 had a trans-configuration in 2 (Figure 2 figurational analysis and NOESY measurements could not discrimina tive configurations for (2S*, 3S*) or (2R*, 3R*). To solve this issue an absolute configuration of 2, the ECD spectra of 2 and its enantiomer w experimental ECD spectrum of 2 showed a good agreement with the ca trum of the 2R, 3R-isomer ( Figure 3). Therefore, the structure of 2 w epimer of 1 and to be a 2R-acetamide-3R-methyl-thiolactone.
Notably, the 1 H and 13 C NMR data in CDCl3 of 2 were almost the s previously reported thiolactone with 2R, 3S-configuration isolated chrysogenum (Table S1 and Figure S15) [20]. The reported compound uration possesses the same planar structure as 2 in this study. In the or compound with 2R, 3S-configuration, by the NOE correlation betwee H-2 (δH 4.45), the authors insisted that the two protons were oriented the ring system. However, its 1D NOE spectrum for the reported com nals from H-3 (δH 2.24) to H-2/H-4/H-5/H-6 and NH, making it uncle orientation of H-3 to the same side of H-2 or not (Figures S15 and S1 reported configuration is correct, H-2 and H-3 are in syn relation, and small scalar coupling constant, but H-2 in the reported thiolactone h constant (12.5 Hz) as in the revised structure (Table S1). In this study pared and checked the selective 1D NOESY data of 2 with those for pound. As noted above, 2 exhibited strong NOE correlations from Hfrom H-4b to H-3 but not from H-4b to H-2, suggesting that H-2 and H face. Furthermore, the reported compound with 2R, 3S-configuration figuration) are enantiomers and should have the same but opposite-in tion values. However, the optical rotation values of the reported thio  Compound 3 was isolated as a white amorphous powder, and its was determined to be C21H32O2. By the comparison of the 1 H and 13 C N ESIMS, and optical rotation data of 3 with those reported previously was identified as a known compound, (+)-03219A, Δ 8,9 -3β-hydroxy-5 [21][22][23]. Compound 3 was isolated as a white amorphous powder, and its molecular formula was determined to be C 21 H 32 O 2 . By the comparison of the 1 H and 13 C NMR (Table 2), HR-ESIMS, and optical rotation data of 3 with those reported previously in the literature, 3 was identified as a known compound, (+)-03219A, ∆ 8,9 -3β-hydroxy-5α-17-acetyl steroid [21][22][23]. Compound 4 was purified as a white amorphous powder, and its molecular formula was determined to be C 21 H 32 O 2 by HR-ESIMS, which is identical to that of 3, with 6 degrees of unsaturation. The 1 H and 13 C NMR data of 4 are summarized in Table 2. The 1 H NMR data for 4 revealed the signals of three methyl groups (δ H 0.57, 0.94, and 2.13), one oxymethine (δ H 3.97), nine methylenes, and three sp 3 methines. The 13 C NMR and HSQC data of 4 exhibited 21 carbon signals containing three methyls (δ C 13.2, 17.3, and 31.7), one oxymethine (δ C 67.2), nine methylenes, two olefinic quaternary carbons (δ C 129.0 and 137.2), two sp 3 quarternary carbons (δ C 37.6 and 45.1), and one ketone carbonyl carbon (δ C 212.5). The planar structure of 4 was elucidated by 1 H-1 H COSY and HMBC data ( Figure 5). (δ C 137.2) indicated a double bond was located at C-8 and C-9. Additionally, the HMBC correlations from H-21 to C-17 (δ C 63.5)/C-20 (δ C 212.5) supported the assignment of an acetyl moiety connected to C-17 of the five-membered ring. The planar structure of 4 was the same as that of 3, (+)-03219A [23], except for the difference in the chemical shifts around the oxymethine (δ H 3.97 and δ C 67.2) at C-3, suggesting that the stereochemistry of C-3 might be different from 3 ( Figure 1 and Table 2). The stereochemistry of 4 was determined by analysis of the ROESY spectrum, 1D NOESY data, coupling constants, and the pyridine-induced deshielding effect. The relative configuration  Figure S27). Furthermore, the small coupling constant of H-3 at δ H 3.97 (t, J = 2.8) was indicative of the C-3 hydroxyl group being axial from an examination of the Dreiding model (Table 2 and Figure 5) [24]. The significant deshielded chemical shifts of H eq -3 (∆δ H = +0.32) and H ax -5 (∆δ H = +0.48) in pyridine-d 5 compared with those in CD 3 OD indicated that OH-3 and H-5 adopted α-orientation, supporting the identified orientation ( Figure 6 and Figure S29) [25][26][27][28]. Consequently, the structure of 4 was determined as a new epimer of 3, ∆ 8,9 -3α-hydroxy-5α-17-acetyl steroid.  Figure S27). Furthermore, the small coupling constant of H-3 at δH 3.97 (t, J = 2.8) was indicative of the C-3 hydroxyl group being axial from an examination of the Dreiding model (Table 2 and Figure 5) [24]. The significant deshielded chemical shifts of Heq-3 (ΔδH = +0.32) and Hax-5 (ΔδH = +0.48) in pyridine-d5 compared with those in CD3OD indicated that OH-3 and H-5 adopted α-orientation, supporting the identified orientation (Figures 6 and S29) [25][26][27][28]. Consequently, the structure of 4 was determined as a new epimer of 3, Δ 8,9 -3α-hydroxy-5α-17-acetyl steroid.  Compound 5 was obtained as a white amorphous powder. The NMR data of 5 were similar to those of 4, except for the absence of signals for the oxymethine at C-3 (δH 3.97 and δC 67.2) in 4 and the appearance of a ketone signal at C-3 (δC 214.6) in 5 ( Table 2), revealing that 5 would be an oxidized form of 4. The 1 H and 13 C NMR spectra, compared to those of 3 and 4, showed the significantly deshielded chemical shifts of C-2 (δH 2.31/2.53 and δC 39.1) and C-4 (δH 2.11/2.40 and δC 45.7). Additionally, the HMBC correlations between H-2b (δH 2.53)/H-4 (δH 2.11/2.40) and C-3 (δC 214.6) determined the position of the ketone at C-3 ( Figure 7). To clearly confirm the structure of 5, 4 was oxidized to obtain the semisynthetic 5. Both 5 and semisynthetic 5 exhibited identical 1 H NMR, HSQC, and HMBC spectra (Figures S35, S36, and S37). The molecular formula of semisynthetic 5 was determined to be C21H30O2 by HR-ESIMS (m/z 337.2134 [M + Na] + , calcd. for C21H30O2Na, 337.2138). Based on these results, the structure of 5 was determined as a 3-keto derivative of 4, with 7 degrees of unsaturation. Therefore, the structures of 5 and semisynthetic 5 were designated as Δ 8,9 -5α-3,20-dione-17-acetyl steroids.  in CD3OD indicated that OH-3 and H-5 adopted α-orient orientation (Figures 6 and S29) [25][26][27][28]. Consequently, the as a new epimer of 3, Δ 8,9 -3α-hydroxy-5α-17-acetyl steroid   Compound 5 was obtained as a white amorphous powder. The NMR data of 5 were similar to those of 4, except for the absence of signals for the oxymethine at C-3 (δ H 3.97 and δ C 67.2) in 4 and the appearance of a ketone signal at C-3 (δ C 214.6) in 5 ( Table 2), revealing that 5 would be an oxidized form of 4. The 1 H and 13 C NMR spectra, compared to those of 3 and 4, showed the significantly deshielded chemical shifts of C-2 (δ H 2.31/2.53 and δ C 39.1) and C-4 (δ H 2.11/2.40 and δ C 45.7). Additionally, the HMBC correlations between H-2b (δ H 2.53)/H-4 (δ H 2.11/2.40) and C-3 (δ C 214.6) determined the position of the ketone at C-3 ( Figure 7). To clearly confirm the structure of 5, 4 was oxidized to obtain the semisynthetic 5. Both 5 and semisynthetic 5 exhibited identical 1 H NMR, HSQC, and HMBC spectra ( Figures S35, S36 and S37). The molecular formula of semisynthetic 5 was determined to be C 21  Based on these results, the structure of 5 was determined as a 3-keto derivative of 4, with 7 degrees of unsaturation. Therefore, the structures of 5 and semisynthetic 5 were designated as ∆ 8,9 -5α-3,20-dione-17-acetyl steroids.

α-Glucosidase Inhibitory Activities of Compounds
Compounds 1-4 were evaluated for α-glucosidase inhibitory activities (Table 3). Compound 4 exhibited the most significant inhibitory effect with an IC 50 value of 86.0 µM, while 2 and 3 showed moderate activities with IC 50 values of 148.4 and 279.7 µM, respectively. Further, 1 exhibited weak inhibitory activity at a concentration of 400 µM. The change in the stereochemistry of the compounds remarkably altered the α-glucosidase inhibitory activities. Compounds 1 and 2, as well as 3 and 4, are stereoisomers of each other. Compounds 2 and 4 showed stronger α-glucosidase inhibitory effects than 1 and 3. It could be noted herein that the stereochemistry was important for α-glucosidase inhibitory activity. ketone at C-3 (Figure 7). To clearly confirm the structure of 5, semisynthetic 5. Both 5 and semisynthetic 5 exhibited iden HMBC spectra (Figures S35, S36, and S37). The molecular for determined to be C21H30O2 by HR-ESIMS (m/z 337.2134 [M + 337.2138). Based on these results, the structure of 5 was deter of 4, with 7 degrees of unsaturation. Therefore, the structur were designated as Δ 8,9 -5α-3,20-dione-17-acetyl steroids.   and 3 (IC 50 = 279.7 µM) demonstrated superior activity as compared to acarbose (IC 50 = 418.9 µM). To the best of our knowledge, this is the first report of new bioactive metabolites with potent α-glucosidase inhibitory activity from the yeast-like fungus Meira. These results show that Meira sp. 1210CH-42 produces unique and diverse metabolites which have the potential for an anti-diabetic agent. The genus Meira is mostly found on land, and secondary metabolites from the marine-derived genus have not yet been reported. Therefore, further research is needed for the marine-derived fungus Meira sp. 1210CH-42 to discover novel secondary metabolites and investigate their biological properties.